CN112980797A - Cell screening model of unmarked melatonin membrane receptor MT1 and application - Google Patents
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Abstract
The invention relates to a construction method and application of a cell screening model of a label-free G protein-coupled receptor (GPCR), in particular to a cell screening model of a label-free melatonin membrane receptor subtype MT 1. The invention establishes a method for screening the agonists and antagonists of the MT1 receptor by using a cell line stably expressed by MT1 based on a label-free cell integration pharmacological technology. This method can also be used to study modulators that affect the downstream pathway of the MT1 receptor. The MT1 cell screening model constructed by the invention does not need fluorescent labeling, does not need an additional indicator in the detection process, and has the characteristics of target spot-path integration response, no damage to cells, reliable detection result, high sensitivity, high screening quantity, simplicity and convenience in operation and the like. It is used for searching agonists, antagonists and pathway modulators of MT1 receptor from natural product library, metabolite library and combinatorial chemistry library, and drug screening of insomnia, abnormal circadian rhythm, mood disorder and tumor-related diseases in which MT1 receptor participates.
Description
Technical Field
The invention relates to a construction method of a cell screening model of a non-labeled GPCR, in particular to a cell screening model of a non-labeled melatonin membrane receptor MT1 and application thereof.
Background
G-protein-coupled receptors (GPCRs) are the most important class of membrane receptors in cell signaling and are one of the most interesting Drug targets in the development of small molecule drugs, and about 34% of modern drugs directly target the receptor family [ Hauser, a. s., et al, Nature Reviews Drug Discovery 2017, 16, 829-842 ]. The melatonin membrane receptor (MT 1) belongs to a member of the GPCR family, and MT1 receptor was first cloned from frogs, sheep and humans in 1994 and consists of 350 amino acids [ Ebisawa T., et al., Proc Natl Acad Sci USA 1994, 91, 6133-; reppert SM., et al, Neuron 1994, 13, 1177-. The MT1 receptor is expressed in the brain, cardiovascular system (including peripheral blood vessels, aorta and heart), immune system, testis, ovary, skin, liver, kidney, adrenal cortex, placenta, breast, retina, pancreas and spleen [ Dubocovich ML., et al, Endocrine 2005, 27, 101-; fischer TW., et al, Exp Dermatol 2008b, 17, 713-; Pandi-Perumal SR., et al, Prog Neurobiol 2008, 85, 335-; slominski a., et al, Endocrine 2005a, 27, 137-; slominski A., et al., Trends Endocrinol Metab 2008, 19, 17-24. Studies have shown that activation of MT1 can lead to vasoconstriction [ Pandi-Perumal SR., et al, Prog Neurobiol 2008, 85, 335-. In addition, studies have shown that the MT1 receptor gene behaves abnormally in addition to mouse phenotype, including impaired sensorimotor gating and pronounced depressed mood, suggesting that the MT1 receptor is important for normal Brain and behavioral function [ Weil ZM., et al, Brain Res Bull 2006, 68, 425-429 ]. Furthermore, studies have shown that the expression of MT1 receptor in breast Cancer cells is directly proportional to the anti-tumor proliferation of melatonin, indicating that MT1 receptor is involved in regulating the proliferation process of breast Cancer [ Collins a., et al., Cancer Lett 2003, 189, 49-57 ]. The construction of a cell screening model of the MT1 receptor for finding out an endogenous ligand, a high-activity agonist, an antagonist and a pathway regulator of the MT1 receptor has great significance for disclosing the biological function and pharmacological characteristics of the MT 1.
At present, the high-throughput screening method for the receptor mainly comprises the traditional radioligand receptor binding assay, GTP γ S binding assay, cyclic adenosine monophosphate (cAMP) assay, calcium flux assay, reporter gene assay, receptor endocytosis assay and β -arrestin recruitment assay [ Bylund, D.B., et al, American Journal of Physiology 1993, 265, L421-L429; harrison, C., et al, Life Sciences 2003, 74, 489-; zhang, r., et al, Acta pharmacolitica Sinica 2012, 33, 372-384; emkey, R., et al, in: Janzen, W.P., et al (Eds.), High Throughput Screening: Methods and protocols, Second Edition; fan, F., et al, Assay and Drug Development Technologies 2007, 5, 127-; zanella, f., et al, Trends in Biotechnology 2010, 28, 237-; luttrell, L.M., et al, Journal of Cell Science 2002, 115, 455-465 ]. However, these methods have certain limitations, for example, the traditional radioligand receptor binding assay requires washing and filtration, the assay cycle is long and the flux is low, and the technology can not distinguish the receptor agonist and antagonist; the other GPCR detection methods mainly aim at the activation of a certain signal path, can not distinguish the activation of a plurality of paths, require fluorescent protein labeling or additional addition of an indicator, are complex to operate, and cause certain damage to cells due to the addition of the indicator, thereby influencing the reliability of a screening result.
Disclosure of Invention
The invention aims to provide a cell screening model of a label-free melatonin membrane receptor MT1, so as to solve the problems in the background technology.
In order to achieve the above objects, a cell screening model of the label-free melatonin membrane receptor MT1 is provided by means of a novel label-free cell integration pharmacological technology to screen the endogenous ligands, agonists, antagonists and pathway modulators of the MT1 receptor, and the drug screening applications of insomnia, abnormal circadian rhythms, mood disorders and tumor-related diseases in which the MT1 receptor participates, with high throughput.
The technical scheme of the invention is as follows:
based on the marker-free cell integrative pharmacological technology, a cell screening model of the MT1 receptor is established by using a cell line CHO-MT1 which stably expresses MT1 and by means of known agonists and antagonists. And judging the agonistic activity and antagonistic activity of the sample to be tested or the regulation influence on a downstream passage according to the similarity of the DMR signal spectrum of the sample to be tested and the DMR characteristic signal spectrum of the known agonist and antagonist.
The label-free cell integration pharmacology technology is characterized in that a Resonance Waveguide Grating (RWG) biosensor is used for converting a dynamic redistribution phenomenon of intracellular components caused by a medicament into an integral and dynamic wavelength shift response signal, the signal is a response value (pm) of wavelength change, and the signal is realized through an Epic optical biosensor 384 micro-porous plate. The establishing process of the model is as follows:
1) CHO-MT1 cells are inoculated in 384 micro-porous plates which are compatible with cells and have optical biosensing function, and the density of the inoculated cells is 1.0-4.5 multiplied by 104The number of the cells per well is 40 muL per well, and the cell culture time after inoculation is 18-24 h.
2) Adding the MT1 receptor agonist melatonin dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with CHO-MT1 cells at the concentration of 0.01-50000 nM, and detecting the DMR characteristic signal spectrum;
3) adding the MT1 receptor antagonist Luzindole dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with CHO-MT1 cells at the concentration of 0.01-100000 nM, and detecting the DMR characteristic signal spectrum;
4) all the obtained DMR characteristic signal spectrums have concentration-response dependence and have sensitivity, saturation and specificity.
Wherein, the screening steps of the sample to be tested with the agonistic activity are as follows:
1) adding the MT1 receptor agonist melatonin dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with CHO-MT1 cells at the concentration of 0.01-50000 nM, and detecting the DMR characteristic signal spectrum;
2) adding a sample to be detected into a micropore plate inoculated with CHO-MT1 cells by 0.01 nM-100 mu M, and detecting a DMR signal spectrum;
3) correlating and analyzing the DMR signal spectrums in the step 1) and the step 2), and if the DMR signal spectrum in the step 2) has contour similarity with the DMR characteristic spectrum in the step 1), carrying out the next step;
4) adding the MT1 receptor antagonist Luzindole into a micropore plate inoculated with CHO-MT1 cells at the concentration of 0.01-100000 nM, pretreating for 5-60 min, adding a sample to be detected with the same concentration as that in the step 2), detecting a DMR signal of the sample, and judging the sample to be an agonist of the MT1 receptor if the DMR signal intensity is lower than that in the step 2).
Wherein, the screening steps of the sample to be tested with antagonistic activity are as follows:
1) respectively adding a sample to be detected and melatonin into a micropore plate inoculated with CHO-MT1 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the melatonin is 0.01-50000 nM, and detecting a DMR signal spectrum;
2) if the sample to be detected in the step 1) does not cause the DMR signal spectrum, continuously adding melatonin with the same concentration as that in the step 1) into the cell plate added with the sample to be detected in the step 1), and detecting the DMR signal spectrum; if the DMR signal is weaker than the melatonin signal in step 1), the test sample can be determined to be an antagonist of MT1 receptor.
Wherein, the step that the sample to be tested has the regulation activity to the MT1 channel is as follows:
1) respectively adding a sample to be detected and an MT1 receptor agonist melatonin into a micropore plate inoculated with CHO-MT1 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the melatonin is 0.01-50000 nM, and detecting a DMR signal spectrum;
2) continuously adding melatonin with the same concentration as that in the step 1) into the cell plate added with the sample to be detected in the step 1), and detecting a DMR signal spectrum for 1-60 min; if the DMR signal is different from the melatonin signal in the step 1) in a certain stage of ascending period, plateau period and delay period;
3) adding an MT1 receptor antagonist Luzindole into a micropore plate inoculated with CHO-MT1 cells at the concentration of 0.1-1000 nM, pretreating for 5-60 min, adding a sample to be detected with the same concentration as that in the step 1), detecting a DMR signal of the sample, and judging that the sample to be detected is a regulator of a downstream signal channel of an MT1 receptor if the DMR signal spectrum is consistent with that of the sample in the step 1).
Wherein the rise period is 1-10 min, the plateau period is 10-20 min and the lag period is 20-60 min.
The cell screening model of the unmarked melatonin membrane receptor MT1 established by the invention can be used for carrying out high-throughput screening on a commercial small molecule library, a self-prepared natural product extract, a component or compound library and a chemical modifier to obtain an agonist, an antagonist and a pathway regulator of the MT1 receptor. In addition, according to the relevance of the target and diseases, the MT1 receptor is found to play an important role in insomnia, abnormal circadian rhythm, mood disorder and tumor, and can also be used for screening drugs for relevant diseases.
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FIG. 1 (A) DMR signature spectra on CHO-MT1 cells at different concentrations of melatonin; (B) concentration-response dependence curves of different concentrations of melatonin on CHO-MT1 cells; wherein the concentration of melatonin is in nM.
FIG. 2 DMR signature spectra of different concentrations of Luzindole on CHO-MT1 cells; where the concentration of Luzindole is in nM.
FIG. 3 (A) DMR signal spectra of fixed concentrations of melatonin after pretreatment of CHO-MT1 cells for 1h with different concentrations of melatonin; (B) after the CHO-MT1 cells are pretreated by melatonin with different concentrations for 1h, a concentration-response dependence curve corresponding to a DMR signal spectrum of the melatonin with fixed concentration is obtained; wherein the concentration of melatonin is in nM.
FIG. 4 (A) DMR signal spectra of fixed concentrations of melatonin after 1h of different concentrations of Luzindole pre-treated CHO-MT1 cells; (B) after CHO-MT1 cells are pretreated by Luzindole with different concentrations for 1h, a concentration-response dependence curve corresponding to a DMR signal spectrum of melatonin with fixed concentration is fixed; wherein the concentration units of melatonin and Luzindole are nM.
Detailed Description
The present invention will now be further described with reference to examples. The examples are given solely for the purpose of illustration and are not intended to be limiting.
Example 1: DMR signature spectra of MT1 receptor agonist melatonin on CHO-MT1 cells
Chinese hamster ovary cells CHO-MT1 cells were obtained from the institute of chemical and physical, university of Chinese academy of sciences, with an inverted microscope from OLYMPUS and melatonin and Luzindole from AMQUAR and Sigma, respectively. The cell culture plate is an Epic optical biosensing 384 micro porous plate purchased from Corning company, the detection platform is a Corning third generation Epic imager, and the detected signal is wavelength shift caused by cell Dynamic Mass Resetting (DMR).
Inoculating CHO-MT1 cells in logarithmic growth phase into a 384-compatible microplate, wherein the inoculation volume of each well is 40 mu L, and the number of the cells inoculated in each well is 1.0 multiplied by 104And (3) placing the inoculated cell plate in a cell culture box for culturing for 20-22 h until the cell fusion degree reaches about 95%, and performing an activity experiment. Changing the cell culture solution in the microporous plate into Hank's balanced salt solution (containing 20 mM HEPES), adding 30 mu L of the cell culture solution into each hole, and placing the cells on an Epic imager to balance for 1h after adding the cells; rescanning the baseline for 2 min, melatonin was added to the plates in a volume of 10 μ L per well at 50000 nM, 16666.67 nM, 5555.56 nM, 1851.85 nM, 617.28 nM, 205.76 nM, 68.59 nM, 22.86 nM, 7.62 nM, 2.54 nM, 0.85 nM, 0.28 nM, 0.09 nM, 0.03 nM and 0.01 nM, 3 replicates, and the DMR signal was monitored in real time on an Epic instrument for 1h, and the EC for melatonin was calculated based on the maximum DMR response of the cells over 60 min after melatonin exposure50The values, results are shown in FIG. 1.
The study shows that melatonin activates MT1 receptor in a dose-dependent manner, the dose response curve is in a single-phase S shape and reaches saturation response, the highest DMR response value reaches 100 pm, and the EC of the melatonin is50The value was 0.21. + -. 0.02. mu.M.
Example 2: DMR characteristic signal profile of MT1 receptor antagonist Luzindole on CHO-MT1 cells
Inoculating CHO-MT1 cells in logarithmic growth phase into a 384-compatible microplate, wherein the inoculation volume of each well is 40 mu L, and the number of the cells inoculated in each well is 1.0 multiplied by 104And (3) placing the inoculated cell plate in a cell culture box for culturing for 20-22 h until the cell fusion degree reaches about 95%, and performing an activity experiment. Changing the cell culture solution in the microporous plate into Hank's balanced salt solution (containing 20 mM HEPES), adding 30 mu L of the cell culture solution into each hole, and placing the cells on an Epic imager to balance for 1h after adding the cells; different concentrations of Luzindole were added to the plates in a volume of 10. mu.L per well at concentrations of 100000 nM, 33333.33 nM, 11111.11 nM, 3703.70 nM, 1234.57 nM, 411.52 nM, 137.17 nM, 45.72 nM, 15.24 nM, 5.08 nM, 1.69 nM, 0.59 nM, 0.19 nM, 0.06 nM and 0.02 nM, rescanning the baseline for 2 min, and the DMR signal was monitored in real time on Epic instruments for 1h in parallel for 3 times, as shown in FIG. 2.
Studies have shown that DMR response signals for different concentrations of Luzindole are close to zero.
Example 3: desensitization DMR signature Spectrum of CHO-MT1 cells
Inoculating CHO-MT1 cells in logarithmic growth phase into a 384-compatible microplate, wherein the inoculation volume of each well is 40 mu L, and the number of the cells inoculated in each well is 1.0 multiplied by 104And (3) placing the inoculated cell plate in a cell culture box for culturing for 20-22 h until the cell fusion degree reaches about 95%, and performing an activity experiment. Changing the cell culture solution in the microporous plate into Hank's balanced salt solution (containing 20 mM HEPES), adding 30 mu L of the cell culture solution into each hole, and placing the cells on an Epic imager to balance for 1h after adding the cells; different concentrations of melatonin were added to the microplate in pre-treated CHO-MT1 cells for 1h, in a volume of 10. mu.L per well, at concentrations of 50000 nM, 16666.67 nM, 5555.56 nM, 1851.85 nM, 617.28 nM, 205.76 nM, 68.59 nM, 22.86 nM, 7.62 nM, 2.54 nM, 0.85 nM, 0.28 nM, 0.09 nM, 0.03 nM and 0.01 nM, in parallel for 3 times; rescanning 2 min baseline, adding melatonin at fixed concentration into microplate, adding volume of 10 μ L and concentration of 200 nM into each well, paralleling for 3 times, monitoring DMR signal for 1h in real time on Epic instrument, and performing melatonin treatment on cellsCalculating IC at the maximum DMR response value within 60 min50The values, results are shown in FIG. 3.
Studies have shown that melatonin dose-dependently desensitizes MT1 receptors, the dose response curve is monophasic "S" type and all reach saturation response, and IC thereof50The value was 0.041. + -. 0.005. mu.M.
Example 4: antagonistic DMR signature profiles of CHO-MT1 cells
Inoculating CHO-MT1 cells in logarithmic growth phase into a 384-compatible microplate, wherein the inoculation volume of each well is 40 mu L, and the number of the cells inoculated in each well is 1.0 multiplied by 104And (3) placing the inoculated cell plate in a cell culture box for culturing for 20-22 h until the cell fusion degree reaches about 95%, and performing an activity experiment. Changing the cell culture solution in the microporous plate into Hank's balanced salt solution (containing 20 mM HEPES), adding 30 mu L of the cell culture solution into each hole, and placing the cells on an Epic imager to balance for 1h after adding the cells; adding MT1 receptor antagonist Luzindole with different concentrations into a microplate for pretreating cells for 1h, wherein the addition volume of each well is 10 muL, and the concentrations are 100000 nM, 33333.33 nM, 11111.11 nM, 3703.70 nM, 1234.57 nM, 411.52 nM, 137.17 nM, 45.72 nM, 15.24 nM, 5.08 nM, 1.69 nM, 0.59 nM, 0.19 nM, 0.06 nM and 0.02 nM, and paralleling for 3 times; rescanning 2 min baseline, adding melatonin with fixed concentration into microplate, adding volume of 10 μ L and concentration of 200 nM into each well, paralleling for 3 times, monitoring DMR signal for 1h in real time on Epic instrument, and calculating IC based on maximum response value of DMR within 60 min of melatonin action50The values, results are shown in FIG. 4.
The study showed that Luzindole antagonizes MT1 receptor in a dose-dependent manner, the dose response curve is monophasic "S" type and all reach saturation response, and the IC is50The value was 2.28. + -. 0.25. mu.M.
The invention establishes an MT1 unmarked screening model based on the unmarked cell integration pharmacological technology, the model has the advantages of no need of fluorescent labeling and no need of adding an indicator in the detection process, and can efficiently and reliably screen a commercialized small molecule library, a self-prepared natural product extract, a component or compound library and a chemical modifier so as to obtain the medicines for treating insomnia, abnormal circadian rhythm, emotional disorder and tumor related diseases regulated and controlled by an MT1 receptor, an MT1 receptor.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A cell screening model of a marker-free melatonin membrane receptor MT1, characterized by: based on the marker-free cell integration pharmacological technology, a cell screening model of the MT1 receptor is established by using a cell line CHO-MT1 which stably expresses MT1 and by means of known agonists and antagonists of the MT1 receptor.
2. The cell screening model of the unlabeled melatonin membrane receptor MT1 of claim 1, wherein: CHO-MT1 cells are inoculated in 384 micro-porous plates which are compatible with cells and have optical biosensing function, and the density of the inoculated cells is 1.0-4.5 multiplied by 104The number of the cells per well is 40 muL per well, and the cell culture time after inoculation is 18-24 h.
3. The cell screening model of the unlabeled melatonin membrane receptor MT1 according to claim 1, wherein: the method specifically comprises the following steps:
[1] adding the MT1 receptor agonist melatonin dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with CHO-MT1 cells at the concentration of 0.01-50000 nM, and detecting the DMR characteristic signal spectrum;
[2] adding the MT1 receptor antagonist Luzindole dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with CHO-MT1 cells at the concentration of 0.01-100000 nM, and detecting the DMR characteristic signal spectrum;
[3] continuously adding melatonin with the lowest concentration corresponding to the highest response intensity into the cell plate added with the melatonin and the Luzindole in the step (1) (2), and respectively detecting a desensitization DMR characteristic signal spectrum and an antagonism DMR characteristic signal spectrum;
[4] all the obtained DMR characteristic signal spectrums have the characteristics of concentration-response dependency relationship, sensitivity, saturation and specificity.
4. The cell screening model of the unlabeled melatonin membrane receptor MT1 of claim 1, wherein the screening step for agonist activity of the test sample is as follows: :
[1] adding the MT1 receptor agonist melatonin dissolved in HBSS buffer salt into a 384 micro-well plate inoculated with CHO-MT1 cells at the concentration of 0.01-50000 nM, and detecting the DMR characteristic signal spectrum;
[2] adding a sample to be detected into a micropore plate inoculated with CHO-MT1 cells by 0.01 nM-100 mu M, and detecting a DMR signal spectrum;
[3] performing correlation analysis on the DMR signal spectrums in the step [1] and the step [2], and if the DMR signal spectrum in the step [2] has contour similarity with the DMR characteristic spectrum in the step [1], performing the next step;
[4] adding the MT1 receptor antagonist Luzindole into a micropore plate inoculated with CHO-MT1 cells at the concentration of 0.01-100000 nM, pretreating for 5-60 min, adding a sample to be detected with the same concentration as that in the step [2], detecting a DMR signal of the sample, and judging the sample to be an agonist of the MT1 receptor if the DMR signal intensity is lower than that in the step [2 ].
5. The model of claim 1, wherein the step of screening for antagonistic activity in a test sample comprises:
[1] respectively adding a sample to be detected and an MT1 receptor agonist melatonin into a micropore plate inoculated with CHO-MT1 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the melatonin is 0.01-50000 nM, and detecting a DMR signal spectrum;
[2] if the sample to be detected in the step (1) does not cause the DMR signal spectrum, continuously adding melatonin with the same concentration as that in the step (1) into the cell plate added with the sample to be detected in the step (1), and detecting the DMR signal spectrum; if the DMR signal is weaker than the melatonin signal in the step [1], the sample to be tested can be judged to be the antagonist of the MT1 receptor.
6. The cell screening model of the unlabeled melatonin membrane receptor MT1 of claim 1, wherein the step of determining the activity of the test sample on the MT1 receptor pathway comprises:
[1] respectively adding a sample to be detected and an MT1 receptor agonist melatonin into a micropore plate inoculated with CHO-MT1 cells, wherein the concentration of the sample to be detected is 0.01 nM-100 mu M, the concentration of the melatonin is 0.01-50000 nM, and detecting a DMR signal spectrum;
[2] continuously adding melatonin with the same concentration as that in the step (1) into the cell plate added with the sample to be detected in the step (1), and detecting a DMR signal spectrum for 1-60 min; if the DMR signal is different from the melatonin signal in the step [1] in a certain stage of ascending period, plateau period and delay period;
[3] adding the MT1 receptor antagonist Luzindole into a micropore plate inoculated with CHO-MT1 cells at the concentration of 0.01-100000 nM, pretreating for 5-60 min, adding the sample to be detected with the same concentration as that in the step [1], detecting the DMR signal of the sample, and judging that the sample to be detected is the regulator of the MT1 receptor downstream signal path if the DMR signal spectrum is consistent with that of the sample in the step [1 ].
7. The model of claim 6, wherein the time period of ascending is 1-10 min, the time period of plateau is 10-20 min, and the time period of retardation is 20-60 min.
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